Method of treating a TL1A-associated disease or disorder with human antibodies to TL1A

ABSTRACT

A fully human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits human TNF-like ligand 1A (hTL1A) is provided. The human anti-hTL1A antibodies are useful in treating diseases or disorders associated with TL1A, such as inflammatory diseases or disorders, e.g., inflammatory bowel diseases, including ulcerative colitis and Crohn&#39;s disease, rheumatoid arthritis, and the like; autoimmune diseases or disorders, such as multiple sclerosis, diabetes, and the like; and allergic reactions, such as asthma and allergic lung inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit ofcurrently pending U.S. patent application Ser. No. 13/291,145, entitled“HUMAN ANTIBODIES TO HUMAN TNF-LIKE LIGAND 1A (TL1A)”, filed Nov. 8,2011, which application claims the benefit under 35 U.S.C §119(e) ofU.S. provisional application Nos. 61/411,276 filed Nov. 8, 2010; and61/478,309 filed Apr. 22, 2011, both of which are herein specificallyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-bindingfragments of human antibodies that specifically bind human TNF-likeligand 1A (hTL1A), and therapeutic methods of using those antibodies.

STATEMENT OF RELATED ART

TL1A is a type II cell membrane protein of the tumor necrosis factorsuperfamily (TNFSF) and also designated as TNFSF15. It is expressed onthe surface of endothelial cells, and activated cells of thehematopoietic lineage, including monocytes, macrophages, lymphocytes,lamina propria mononuclear cells, dendritic cells and plasma cells (Tan,K. B. et al., 1997, Gene 204:35-46; Prehn, J. L. et al., 2007, J Immunol178:4033-4038). It is also expressed in kidney, lung, prostate andthymus (Tan et al., 1997, supra). In endothelial cells, expression ofTL1A is upregulated by IL-1α and TNFα (Migone, T. S. et al., 2002,Immunity 16:479-492). In human fresh blood monocytes andmonocyte-derived dendritic cells, TL1A expression is upregulated byFcγR-mediated or Toll-like receptor (TLR) signaling (Prehn et al., 2007,supra; Meylan, F. et al., 2008, Immunity 29:79-89). TL1A can be cleavedfrom the cell membrane via a mechanism analogous to TNFα and a solubleectodomain form of TL1A has been reported (Migone et al., 2002, supra;Kim, S. et al., 2005, J Immunol Methods 298:1-8; Yang, C. R. et al.,2004, Cancer Res 64:1122-1129). Protein sequencing has confirmed thatthis form of TL1A is liberated following cleavage of themembrane-anchored precursor between residues Ala-71 and Leu-72 (Migoneet al., 2002, supra). Two variant cDNAs that potentially encodeN-terminally truncated versions of TL1A have been identified: VEGI-174(or TL1) (Zhai, Y. et al., 1999, FASEB J 13:181-189) and VEGI-192 (Chew,L. J. et al., 2002, FASEB J 16:742-744). The published data suggest thebiologically active products of the TL1A gene are the full-length typeII transmembrane protein (residues 1-251) and its proteolyticallycleaved ectodomain (residues 72-251) (Migone et al., 2002, supra; Jin etal., 2007, Biochem Biophys Res Commun 364:1-6). A variant of hTL1A,designated as “Fhm”, containing a single amino acid substitution ofGln-167 with Arg, is disclosed in U.S. Pat. No. 6,521,422.

TL1A mediates signals via its cognate receptor Death Receptor 3 (DR3;also known as TNFRSF25; the nucleic acid and amino acid sequences of SEQID NO:251 and 252, respectively), resulting in promoting cell survivaland secretion of pro-inflammatory cytokines, or promoting apoptosis, ina context-dependent manner. TL1A is one of three known ligands (inaddition to FasL and LIGHT) that are bound by the endogenous solubledecoy receptor, DcR3 (also known as TR6, NTR3 or TNFRSF21; the nucleicacid and amino acid sequences of SEQ ID NO:253 and 254, respectively)(Migone et al., 2002, supra; Yang C. R. et al., 2004, Cancer Res64:1122-1129).

DR3 is a TNF receptor-related death-domain receptor expressed on themajority of activated T lymphocytes and NK cells (Migone et al., 2002,supra; Screaton G. R. et al., 1997, Proc Natl Acad Sci (USA)94:4615-4619). TL1A engages DR3 on T cells, enhancing theirresponsiveness to IL-2 (Migone et al., 2002, supra), potentiating T cellproliferation and release of IFNγ and GM-CSF under conditions ofsuboptimal costimulation (Migone et al., 2002, supra; Meylan et al.,2008, supra). TL1A has also been shown to synergize with suboptimallevels of IL-12/IL-18 to induce IFNγ production by CD4⁺ T cells(Papadakis, K. A. et al., 2004, J Immunol 172:7002-7007; Prehn, J. L. etal., 2004, Clin Immunol 112:66-77; Papadakis, K. A. et al., 2005, JImmunol 174:4985-4990; Cassatella, M. A. et al., 2007, J Immunol178:7325-7333).

TL1A has been implicated in various inflammatory diseases and/or autoimmune diseases, including inflammatory bowel diseases [e.g., ulcerativecolitis (UC) and Crohn's disease (CD)], rheumatoid arthritis, multiplesclerosis (MS), atherosclerosis, and the like (see Bayry, J., 2010,Nature Reviews/Rheumatology 6:67-68; Takedatsu, H. et al., 2008,Gastroenterology 135:552-567; Prehn et al., 2004, supra; Bamias, G. etal., 2008, Clin Immunol 129:249-255; Bull, M. J. et al., 2008, J Exp Med205:2457-2464; Pappu, B. P. et al., 2008, J Exp Med 205:1049-1062;Bamias, G. et al., 2003, J Immunol 171:4868-4874; Kang, Y. et al., 2005,Cytokine 29:229-235). Although the majority of the published data areconsistent with a pivotal role for TL1A in driving differentiation ofT_(H)1 and T_(H)17 effector function, a recent study has proposed a rolefor the TL1A/DR3 interaction in development of T_(H)2 T cell responsesin asthma models (Fang, L. et al., 2008, J Exp Med 205:1037-1048). Thus,the use of TL1A inhibitors, such as fully human antibodies against TL1Awith high affinities and neutralizing activity, alone or in combinationwith currently available anti-inflammatory agents, immunosuppresants(e.g., TNF-α antagonists, cortisone or steroids, and the like), and/oranti-allergy agents, provides effective treatment for these diseases anddisorders.

The nucleic acid and the amino acid sequences of human TL1A are shown inSEQ ID NOS: 243 and 244, respectively, and those of Fhm are shown in SEQID NOS:245 and 246, respectively. Antibodies to TL1A are disclosed in,for example, U.S. Pat. No. 7,597,886, U.S. Pat. No. 7,820,798 and US2009/0280116.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides fully human monoclonalantibodies (mAbs) and antigen-binding fragments thereof thatspecifically bind and neutralize human TL1A (hTL1A) activity.

The antibodies can be full-length (for example, an IgG1 or IgG4antibody) or may comprise only an antigen-binding portion (for example,a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

In one embodiment, the invention features an antibody or antigen-bindingfragment of an antibody comprising a heavy chain variable region (HCVR)selected from the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 82,98, 114, 118, 134, 138, 154, 158, 174, 178, 194, 198, 214, 218 and 234,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity. In anotherembodiment, the antibody or an antigen-binding fragment thereofcomprises a HCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, 18, 34, 50, 66, 134, 174 and 234. In yetanother embodiment, the antibody or fragment thereof comprises a HCVRcomprising SEQ ID NO:2, 18, 174 or 234.

In one embodiment, the antibody or fragment thereof further comprises alight chain variable region (LCVR) selected from the group consisting ofSEQ ID NO:10, 26, 42, 58, 74, 90, 106, 116, 126, 136, 146, 156, 166,176, 186, 196, 206, 216, 226 and 236, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity. In another embodiment, the antibody orantigen-binding portion of an antibody comprises a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO:10, 26,42, 58, 74, 136, 176 and 236. In yet another embodiment, the antibody orfragment thereof comprises a LCVR comprising SEQ ID NO:10, 26, 176 or236.

In further embodiments, the antibody or fragment thereof comprises aHCVR and LCVR (HCVR/LCVR) sequence pair selected from the groupconsisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106,114/116, 118/126, 134/136, 138/146, 154/156, 158/166, 174/176, 178/186,194/196, 198/206, 214/216, 218/226 and 234/236. In one embodiment, theantibody or fragment thereof comprises a HCVR and LCVR selected from theamino acid sequence pairs of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74,134/136, 174/176 and 234/236. In another embodiment, the antibody orfragment thereof comprises a HCVR/LCVR pair comprising SEQ ID NO:2/10,18/26, 174/176 or 234/236.

In a second aspect, the invention features an antibody orantigen-binding fragment of an antibody comprising a heavy chaincomplementarity determining region 3 (HCDR3) amino acid sequenceselected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88,104, 124, 144, 164, 184, 204 and 224, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a light chain CDR3 (LCDR3) amino acidsequence selected from the group consisting of SEQ ID NO:16, 32, 48, 64,80, 96, 112, 132, 152, 172, 192, 212 and 232, or substantially similarsequences thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity. In one embodiment, the antibody or fragmentthereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQID NO:8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 124/132,144/152, 164/172, 184/192, 204/212 or 224/232. In another embodiment,the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acidsequence pair comprising SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,124/132, 164/172 or 224/232. In yet another embodiment, the antibody orfragment thereof comprises a HCDR3/LCDR3 amino acid sequence paircomprising SEQ ID NO:8/16, 24/32, 164/172 or 224/232.

In a further embodiment, the invention features an antibody or fragmentthereof further comprising a heavy chain CDR1 (HCDR1) amino acidsequence selected from the group consisting of SEQ ID NO:4, 20, 36, 52,68, 84, 100, 120, 140, 160, 180, 200 and 220, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a heavy chain CDR2 (HCDR2) amino acidsequence selected from the group consisting of SEQ ID NO:6, 22, 38, 54,70, 86, 102, 122, 142, 162, 182, 202 and 222, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and/or a light chain CDR1 (LCDR1) aminoacid sequence selected from the group consisting of SEQ ID NO:12, 28,44, 60, 76, 92, 108, 128, 148, 168, 188, 208 and 228, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and/or a light chain CDR2 (LCDR2)amino acid sequence selected from the group consisting of SEQ ID NO:14,30, 46, 62, 78, 94, 110, 130, 150, 170, 190, 210 and 230, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity. In one embodiment,the antibody or fragment thereof comprises a combination ofHCDR1/HCDR2/HCDR3 selected from the group consisting of SEQ ID NO:4/6/8,20/22/24, 36/38/40, 52/54/56, 68/70/72, 84/86/88, 100/102/104,120/122/124, 140/142/144, 160/162/164, 180/182/184, 200/202/204 and220/222/224; and/or a combination of LCDR1/LCDR2/LCDR3 selected from thegroup consisting of 6SEQ ID NO:12/14/16, 28/30/32, 44/46/48, 60/62/64,76/78/80, 92/94/96, 108/110/112, 128/130/132, 148/150/152, 168/170/172,188/190/192, 208/210/212 and 228/230/232. In another embodiment, theheavy and light chain CDR amino acid sequences comprise a CDR sequencecombination selected from the group consisting of SEQ IDNO:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48,52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96,100/102/104/108/110/112, 120/122/124/128/130/132,140/142/144/148/150/152, 160/162/164/168/170/172,180/182/184/188/190/192, 200/202/204/208/210/212 and220/222/224/228/230/232. In another embodiment, the antibody orantigen-binding fragment thereof comprises heavy and light chain CDRsequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32,36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80,120/122/124/128/130/132, 160/162/164/168/170/172 or220/222/224/228/230/232. In yet another embodiment, the heavy and lightchain CDR amino acid sequences comprise a CDR sequence combination ofSEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 160/162/164/168/170/172 or220/222/224/228/230/232.

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically binds hTL1A,wherein the antibody or fragment thereof comprises heavy and light chainCDR domains contained within heavy and light chain sequence pairsselected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/116, 118/126, 134/136, 138/146,154/156, 158/166, 174/176, 178/186, 194/196, 198/206, 214/216, 218/226and 234/236. Methods and techniques for identifying CDRs within HCVR andLCVR amino acid sequences are known in the art and can be applied toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Conventional definitions that can be applied toidentify the boundaries of CDRs include the Kabat definition, theChothia definition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody. In one embodiment, theantibody or fragment thereof comprises CDR sequences contained within aHCVR and LCVR pair selected from the group consisting of the amino acidsequence pairs of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 134/136,174/176 and 234/236. In another embodiment, the antibody or fragmentthereof comprises CDR sequences contained within the HCVR and LCVRsequence pair of SEQ ID NO: 2/10, 18/26, 174/176 or 234/236.

In another related embodiment, the invention provides an antibody orantigen-binding fragment thereof that competes for specific binding tohTL1A with an antibody or antigen-binding fragment comprising heavy andlight chain CDR sequences of SEQ ID NO:4/6/8/12/14/16,20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64,68/70/72/76/78/80, 120/122/124/128/130/132, 160/162/164/168/170/172 or220/222/224/228/230/232. In one embodiment, the antibody orantigen-binding fragment thereof competes for specific binding to hTL1Awith an antibody or antigen-binding fragment comprising heavy and lightchain CDR sequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32,160/162/164/168/170/172 or 220/222/224/228/230/232. In anotherembodiment, the antibody or antigen-binding fragment of the inventioncompetes for specific binding to hTL1A with an antibody orantigen-binding fragment comprising a HCVR/LCVR sequence pair of SEQ IDNO:2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 or 234/236. In yetanother embodiment, the antibody or antigen-binding fragment thereofcompetes for specific binding to hTL1A with an antibody orantigen-binding fragment comprising a HCVR/LCVR sequence pair of SEQ IDNO:2/10, 18/26, 174/176 or 234/236.

In another related embodiment, the invention provides an antibody orantigen-binding fragment thereof that binds the same epitope on hTL1Athat is recognized by an antibody or fragment thereof comprising heavyand light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16,20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64,68/70/72/76/78/80, 120/122/124/128/130/132, 160/162/164/168/170/172 or220/222/224/228/230/232. In one embodiment, the antibody orantigen-binding fragment thereof binds the same epitope on hTL1A that isrecognized by an antibody or antigen-binding fragment thereof comprisingheavy and light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16,20/22/24/28/30/32, 160/162/164/168/170/172 or 220/222/224/228/230/232.In another embodiment, the antibody or antigen-binding fragment of theinvention recognizes the same epitope on hTL1A that is recognized by anantibody or antigen-binding fragment thereof comprising a HCVR/LCVRsequence pair of SEQ ID NO: SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74,134/136, 174/176 or 234/236. In yet another embodiment, the antibody orantigen-binding fragment thereof recognizes the same epitope on hTL1Athat is recognized by an antibody or antigen-biniding fragment thereofcomprising a HCVR/LCVR sequence pair of SEQ ID NO:2/10, 18/26, 174/176or 234/236.

In a third aspect, the invention provides nucleic acid moleculesencoding anti-TL1A antibodies or fragments thereof described above.Recombinant expression vectors carrying the nucleic acids of theinvention, and isolated host cells, e.g., bacterial cells, such as E.coli, or mammalian cells, such as CHO cells, into which such vectorshave been introduced, are also encompassed by the invention, as aremethods of producing the antibodies by culturing the host cells underconditions permitting production of the antibodies, and recovering theantibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1, 17, 33, 49, 65, 81, 97, 113,117, 133, 137, 153, 157, 173, 177, 193, 197, 213, 217 and 233, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof. In another embodiment, theantibody or fragment thereof comprises a HCVR encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO:1, 17, 33, 49,65, 133, 173 and 233. In yet another embodiment, the antibody orfragment thereof comprises a HCVR encoded by the nucleic acid sequenceof SEQ ID NO:1, 17, 173 or 233.

In one embodiment, the antibody or fragment thereof further comprises aLCVR encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:9, 25, 41, 57, 73, 89, 105, 115, 125, 135, 145,155, 165, 175, 185, 195, 205, 215, 225 and 235, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof. In another embodiment, the antibody orfragment thereof comprises a LCVR encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 135,175 and 235. In yet another embodiment, the antibody or fragment thereofcomprises a LCVR encoded by the nucleic acid sequence of SEQ ID NO:9,25, 175 or 235.

In further embodiments, the antibody or fragment thereof comprises aHCVR and LCVR (HCVR/LCVR) sequence pair encoded by a nucleic acidsequence pair selected from the group consisting of SEQ ID NO:1/9,17/25, 33/41, 49/57, 65/73, 81/89, 97/105, 113/115, 117/125, 133/135,137/145, 153/155, 157/165, 173/175, 177/185, 193/195, 197/205, 213/215,217/225 and 233/235. In one embodiment, the antibody or fragment thereofcomprises a HCVR/LCVR sequence pair encoded by a nucleic acid sequencepair selected from the group consisting of SEQ ID NO:1/9, 17/25, 33/41,49/57, 65/73, 133/135, 173/175 and 233/235. In yet another embodiment,the antibody or fragment thereof comprises a HCVR/LCVR pair encoded by anucleic acid sequence pair of SEQ ID NO:1/9, 17/25, 173/175 or 233/235.

In one embodiment, the invention features an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:7,23, 39, 55, 71, 87, 103, 123, 143, 163, 183, 203 and 223, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof; and a LCDR3 domain encodedby a nucleotide sequence selected from the group consisting of SEQ IDNO:15, 31, 47, 63, 79, 95, 111, 131, 151, 171, 191, 211 and 231, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof. In another embodiment, theantibody or fragment thereof comprises a HCDR3 and LCDR3 sequence pairencoded by the nucleic acid sequence pair of SEQ ID NO:7/15, 23/31,39/47, 55/63, 71/79, 87/95, 103/111, 123/131, 143/151, 163/171, 183/191,203/211 or 223/231. In another embodiment, the antibody or fragmentthereof comprises a HCDR3 and LCDR3 sequence pair encoded by the nucleicacid sequence pair of SEQ ID NO:7/15, 23/31, 39/47, 55/63, 71/79,123/131, 163/171 or 223/231. In yet another embodiment, the HCDR3/LCDR3sequence pair is encoded by the nucleic acid sequence pair of SEQ IDNO:7/15, 23/31, 163/171 or 223/231.

In a further embodiment, the antibody or fragment thereof furthercomprises, a HCDR1 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO:3, 19, 35, 51, 67, 83, 99, 119, 139,159, 179, 199 and 219, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof;a HCDR2 domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:5, 21, 37, 53, 69, 85, 101, 121, 141, 161, 181,201 and 221, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof; a LCDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:11, 27, 43, 59, 75, 91, 107, 127, 147, 167, 187,207 and 227, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof; and aLCDR2 domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 129, 149, 169, 189,209 and 229, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof. In oneembodiment, the antibody or fragment thereof comprises a combination ofHCDR1/HCDR2/HCDR3 encoded by SEQ ID NO:3/5/7, 19/21/23, 35/37/39,51/53/55, 67/69/71, 83/85/87, 99/101/103, 119/121/123, 139/141/143,159/161/163, 179/181/183, 199/201/203 or 219/221/223; and a combinationof LCDR1/LCDR2/LCDR3 encoded by SEQ ID NO:11/13/15, 27/29/31, 43/45/47,59/61/63, 75/77/79, 91/93/95, 107/109/111, 127/129/131, 147/149/151,167/169/171, 187/189/191, 207/209/211 or 227/229/231. In one embodiment,the antibody or fragment thereof comprises heavy and light chain CDRsequences encoded by a nucleic acid sequence combination selected fromthe group consisting of SEQ ID NO:3/5/7/11/13/15, 19/21/23/27/29/31,35/37/39/43/45/47, 51/53/55/59/61/63, 67/69/71/75/77/79,83/85/87/91/93/95, 99/101/103/107/109/111, 119/121/123/127/129/131,139/141/143/147/149/151, 159/161/163/167/169/171,179/181/183/187/189/191, 199/201/203/207/209/211 and219/221/223/227/229/231. In another embodiment, the antibody orantigen-binding portion thereof comprises heavy and light chain CDRsequences encoded by a nucleic acid sequence combination of SEQ IDNO:3/5/7/11/13/15, 19/21/23/27/29/31, 35/37/39/43/45/47,51/53/55/59/61/63, 67/69/71/75/77/79, 119/121/123/127/129/131,159/161/163/167/169/171 or 219/221/223/227/229/231. In yet anotherembodiment, the antibody or antigen-binding portion thereof comprisesheavy and light chain CDR sequences encoded by a nucleic acid sequencecombination of SEQ ID NO: 3/5/7/11/13/15, 19/21/23/27/29/31,159/161/163/167/169/171 or 219/221/223/227/229/231.

In a fourth aspect, the invention features an isolated antibody orantigen-binding fragment of an antibody that specifically binds hTL1A,comprising a HCDR3 and a LCDR3, wherein the HCDR3 comprises an aminoacid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶ (SEQ ID NO:239),wherein X¹ is Thr or Ala, X² is Lys, Arg or absent, X³ is Glu, Gly orabsent, X⁴ is Asp, Pro or absent, X⁵ is Leu or absent, X⁶ is Arg, Tyr,Glu or absent, X⁷ is Gly, Asp, Ala or absent, X⁹ is Asp, Ser or Tyr, X⁹is Tyr or Trp, X¹⁰ is Tyr or Asp, X¹¹ is Tyr, Lys or Ile, X¹² is Gly,Tyr, Asn, or Ser, X¹³ is Val, Gly or Ser, X¹⁴ is Phe or Met, X¹⁵ is Asp,and X¹⁶ is Tyr or Val; and the LCDR3 comprises an amino acid sequence ofthe formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:242), wherein X¹ isGln, X² is Gln, X³ is Tyr, Leu or Phe, X⁴ is His, Tyr or Asn, X⁵ is Argor Ser, X⁶ is Ser, Thr or Tyr, X⁷ is Trp or Pro, X⁹ is Phe, Leu orabsent, and X⁹ is Thr.

In a further embodiment, the antibody or fragment thereof furthercomprises a HCDR1 sequence comprising an amino acid sequence of theformula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:237), wherein X¹ is Gly, X²is Phe, X³ is Thr, X⁴ is Phe, X⁵ is Ser, X⁶ is Thr, Ser or Asn, X⁷ isTyr, and X⁸ is Gly, Trp, Val or Ala; a HCDR2 sequence comprising anamino acid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ IDNO:238), wherein X¹ is Ile or Val, X² is Ser or Lys, X³ is Gly or Glu,X⁴ is Thr, Asp, Ser or Arg, X⁵ is Gly, X⁶ is Arg, Ser or Gly, X⁷ is Thr,Glu or Ser, and X⁸ is Thr or Lys; a LCDR1 sequence comprising an aminoacid sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQID NO:240), wherein X¹ is Gln, X² is Thr, Ser, Ala or Gly, X³ is Ile, X⁴is Ser or Leu, X⁵ is Tyr or absent, X⁶ is Ser or absent, X⁷ is Ser orabsent, X⁸ is Asn or absent, X⁹ is Asn or absent, X¹⁰ is Lys or absent,X¹¹ is Ser, Asn or Thr, and X¹² is Trp or Tyr; and a LCDR2 sequencecomprising an amino acid sequence of the formula X¹-X²-X³ (SEQ IDNO:241) wherein X¹ is Ala, Trp or Ser, X² is Ala or Thr, and X³ is Ser.

In a fifth aspect, the invention features a human anti-TL1A antibody orantigen-binding fragment thereof comprising a heavy chain variableregion (HCVR) encoded by nucleotide sequence segments derived fromV_(H), D_(H) and J_(H) germline sequences, and a light chain variableregion (LCVR) encoded by nucleotide sequence segments derived from V_(K)and J_(K) germline sequences. In certain embodiments, the antibody orantigen-binding fragment thereof comprises the HCVR and the LCVR encodedby nucleotide sequence segments derived from a germline gene combinationselected from the group consisting of: (i) V_(H)3-23, D_(H)2-21, J_(H)4,V_(K)1-5 and J_(K)1; (ii) V_(H)3-7, D_(H)1-7, J_(H)6, V_(K)4-1 andJ_(K)3; (iii) V_(H)3-23, D_(H)2-2, J_(H)6, V_(K)1-9 and J_(K)2; (iv)V_(H)3-23, D_(H)6-6, J_(H)4, V_(K)1-9 and J_(K)4; (v) V_(H)1-2,D_(H)2-15, J_(H)3, V_(K)1-12 and J_(K)4; (vi) V_(H)4-34, D_(H)3-9,J_(H)4, V_(K)3-20 and J_(K)4; (vii) V_(H)4-34, D_(H)1-1, J_(H)4,V_(K)3-20 and J_(K)4; and (viii) V_(H)4-34, D_(H)3-3, J_(H)4, V_(K)2-24and J_(K)4.

In a sixth aspect, the invention features an antibody or antigen-bindingfragment thereof that specifically binds to hTL1A or Fhm with anequilibrium dissociation constant (K_(D)) of about 1 nM or less, asmeasured by surface plasmon resonance assay (for example, BIACORE™). Incertain embodiments, the antibody of the invention exhibits a K_(D) ofabout 800 pM or less; about 700 pM or less; about 600 pM or less; about500 pM or less; about 400 pM or less; about 300 pM or less; about 200 pMor less; about 150 pM or less; about 100 pM or less; about 90 pM orless; about 80 pM or less; about 50 pM or less; or 30 pM or less.

In a seventh aspect, the present invention provides an anti-hTL1Aantibody or antigen-binding fragment thereof that binds hTL1A protein ofSEQ ID NO:244, but does not cross-react with a variant thereof, such asFhm of SEQ ID NO:246, as determined by, for example, ELISA, surfaceplasmon resonance assay, or Luminex® xMAP® Technology, as describedherein. Fhm contains a single amino acid substitution at position 167,corresponding to Gln in hTL1A, with Arg (see U.S. Pat. No. 6,521,422).In related embodiments, the invention also provides an anti-hTL1Aantibody or antigen-binding fragment thereof that binds a hTL1A proteinand cross-reacts with an Fhm. In another related embodiment, theinvention provides an anti-hTL1A antibody or antigen binding fragmentthereof that does not cross-react with mouse TL1A (mTL1A: SEQ ID NO:250,encoded by the nucleotide sequence of SEQ ID NO:249) but doescross-react with TL1A of cynomolgus monkey (Macaca fascicularis, orMfTL1A: SEQ ID NO:248, encoded by the nucleotide sequence of SEQ IDNO:247) or rhesus monkey (Macaca mulatta: the same amino acid sequenceas MfTL1A). In further related embodiments, the invention provides ananti-hTL1A antibody or antigen-binding fragment thereof thatcross-reacts with both mTL1A and MfTL1A.

The invention encompasses anti-hTL1A antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or e.g., removal of afucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, removal of N-glycosylation site may reduce undesirableimmune reactions against the therapeutic antibodies, or increaseaffinities of the antibodies. In yet other applications, modification ofgalactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In an eighth aspect, the invention features a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds hTL1A and a pharmaceutically acceptable carrier. Inone embodiment, the invention features a composition which is acombination of an antibody or antigen-binding fragment thereof of theinvention, and a second therapeutic agent. The second therapeutic agentmay be one or more of any agent such as immunosuppressants,anti-inflammatory agents, analgesic agents, anti-allergy agents, and thelike, many of which may have overlapping therapeutic effects of oneanother. Suitable immunosuppressants to be used in combination with theanti-hTL1A antibodies of the invention include, but are not limited to,glucocorticoids, cyclosporin, methotrexate, interferon β (IFN-β),tacrolimus, sirolimus, azathioprine, mercaptopurine, opioids,mycophenolate, TNF-binding proteins, such as infliximab, eternacept,adalimumab, and the like, cytotoxic antibiotics, such as dactinomycin,anthracyclines, mitomycin C, bleomycin, mithramycin, and the like,antibodies targeting immune cells, such as anti-CD20 antibodies,anti-CD3 antibodies, and the like. Suitable anti-inflammatory agentsand/or analgesics for combination therapies with anti-hTL1A antibodiesinclude, corticosteroids, non-steroidal anti-inflammatory drugs(NSAIDs), such as aspirin, ibuprofen, naproxen, Cox-2 inhibitors, andthe like, TNF-α antagonists, IL-1 antagonists, IL-6 antagonists,acetaminophen, morphinomimetics, and the like. Suitable anti-allergyagents include antihistamines, glucocorticoids, epinephrine(adrenaline), theophylline, cromolyn sodium and anti-leukotrienes, aswell as anti-cholinergics, decongestants, mast cell stabilizers, and thelike.

In a ninth aspect, the invention features methods for inhibiting hTL1Aactivity using the anti-hTL1A antibody or antigen-binding portion of theantibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment of anantibody of the invention and, optionally, one or more additionaltherapeutic agents described above. The disease or disorder treated isany disease or condition which is improved, ameliorated, inhibited orprevented, or its occurrence rate reduced compared to that withoutanti-hTL1A antibody treatment, by removal, inhibition or reduction ofTL1A activity. Examples of diseases or disorders treatable by themethods of the invention include, but are not limited to, inflammatorydiseases and/or autoimmune diseases, such as inflammatory bowel diseases(IBD) including UC and CD, RA, MS, type 1 and type 2 diabetes,psoriasis, psoriatic arthritis, ankylosing spondylitis, atopicdermatitis, and the like; allergic reactions or conditions, includingasthma, allergic lung inflammation, and the like; cancersatherosclerosis, infections, neurodegenerative diseases, graftrejection, graft vs. host diseases (GVHD), cardiovasculardisorders/diseases, and the like.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present invention is described in detail, it is to beunderstood that this invention is not limited to particular methods, andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

The term “human TNF-like ligand 1A” or “hTL1A”, as used herein, refersto hTL1A having the nucleic acid sequence shown in SEQ ID NO:243 and theamino acid sequence of SEQ ID NO:244, or a biologically active fragmentthereof, as well as hTL1A variants, including Fhm having the nucleicacid sequence shown in SEQ ID NO:245 and the amino acid sequence of SEQID NO:246, or a biologically active fragment thereof, unlessspecifically indicated otherwise.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region (HCVR)and a heavy chain constant region (C_(H); comprised of domains C_(H)1,C_(H)2 and C_(H)3). Each light chain is comprised of a light chainvariable region (LCVR) and a light chain constant region (C_(L)). TheHCVR and LCVR can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example, residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences.

The fully-human anti-TL1A antibodies disclosed herein may comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The present inventionincludes antibodies, and antigen-binding fragments thereof, which arederived from any of the amino acid sequences disclosed herein, whereinone or more amino acids within one or more framework and/or CDR regionsare mutated to the corresponding residue(s) of the germline sequencefrom which the antibody was derived, or to the corresponding residue(s)of another human germline sequence, or to a conservative amino acidsubstitution of the corresponding germline residues(s) (such sequencechanges are referred to herein collectively as “germline mutations”). Aperson of ordinary skill in the art, starting with the heavy and lightchain variable region sequences disclosed herein, can easily producenumerous antibodies and antigen-binding fragments which comprise one ormore individual germline back-mutations or combinations thereof. Incertain embodiments, all of the framework and/or CDR residues within theV_(H) and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residues of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-TL1A antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-TL1A antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, 2 or 1, conservative amino acidsubstitution(s) relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

Unless specifically indicated otherwise, the term “antibody” (Ab), asused herein, shall be understood to encompass antibody moleculescomprising two immunoglobulin heavy chains and two immunoglobulin lightchains (i.e., “full antibody molecules”) as well as antigen-bindingfragments thereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-displayantibody libraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2, (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

In certain embodiments, antibody or antibody fragments of the inventionmay be conjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiological conditions. Specific binding canbe characterized by an equilibrium dissociation constant (K_(D)) ofabout 3000 nM or less (i.e., a smaller K_(D) denotes a tighter binding),about 2000 nM or less, about 1000 nM or less; about 500 nM or less;about 300 nM or less; about 200 nM or less; about 100 nM or less; about50 nM or less; about 1 nM or less; or about 0.5 nM or less. Methods fordetermining whether two molecules specifically bind are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. An isolated antibody that specifically bindshTL1A may, however, exhibit cross-reactivity to other antigens, such asTL1A molecules from other species, for example, cynomolgus monkey TL1A(SEQ ID NO:248), and/or mouse TL1A (SEQ ID NO:250), and/or a TL1Avariant, such as Fhm (SEQ ID NO:246). Moreover, multi-specificantibodies (e.g., bispecifics) that bind to hTL1A and one or moreadditional antigens are nonetheless considered antibodies that“specifically bind’ hTL1A, as used herein.

The term “high affinity” antibody refers to those antibodies having abinding affinity to hTL1A, expressed as K_(D), of about 1×10⁻⁹ M orless, about 0.5×10⁻⁹ M or less, about 0.25×10⁻⁹ M or less, about 1×10⁻¹⁰M or less, or about 0.5×10⁻¹⁰ M or less, as measured by surface plasmonresonance, e.g., BIACORE™ or solution-affinity ELISA.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

By the term “slow off rate”, “Koff” or “k_(d)” is meant an antibody thatdissociates from hTL1A with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

By the term “intrinsic affinity constant” or “k_(a)” is meant anantibody that associates with hTL1A at a rate constant of about 1×10³M⁻¹s⁻¹ or higher, as determined by surface plasmon resonance, e.g.,BIACORE™.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other mAbs having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTL1A is substantially free of Abs that specifically bind antigensother than hTL1A). An isolated antibody that specifically binds hTL1Amay, however, have cross-reactivity to other antigens, such as TL1Amolecules from other species, such as cynomolgus monkey and mouse,and/or hTL1A variants, such as Fhm.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralizes TL1A activity”), is intended to refer to an antibody whosebinding to TL1A results in inhibition of at least one biologicalactivity of TL1A. This inhibition of the biological activity of TL1A canbe assessed by measuring one or more indicators of TL1A biologicalactivity by one or more of several standard in vitro or in vivo assaysknown in the art (also see examples below).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.A “conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent or degree of similarity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, which is herein incorporated by reference. Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartate andglutamate, and 7) sulfur-containing side chains: cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443 45, herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson, 2000, supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403 410 and, 1997,Nucleic Acids Res. 25:3389 402, each of which is herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, the age and the sizeof a subject treated, the route of administration, and the like, andwill be ascertainable by one skilled in the art using known techniques(see, for example, Lloyd (1999) The Art, Science and Technology ofPharmaceutical Compounding).

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind toTL1A.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to TL1A areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, and the like.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² M throughabout 10⁻⁹ M, when measured by binding to antigen either immobilized onsolid phase or in solution phase. The mouse constant regions arereplaced with desired human constant regions, for example, wild-typeIgG1 (SEQ ID NO:255) or IgG4 (SEQ ID NO:256), or modified IgG1 or IgG4(for example, IgG4 with Ser-108 substituted with Pro as shown in SEQ IDNO:257), to generate the fully human antibodies of the invention. Whilethe constant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics of theantibodies reside in the variable region.

Epitope Mapping and Related Technologies

To screen for antibodies that bind to a particular epitope, a routinecross-blocking assay such as that described in Antibodies, Harlow andLane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed.Other methods include alanine scanning mutants, peptide blots (Reineke,2004, Methods Mol Biol 248:443-63) (herein specifically incorporated byreference in its entirety), or peptide cleavage analysis. In addition,methods such as epitope excision, epitope extraction and chemicalmodification of antigens can be employed (Tomer, 2000, Protein Science9: 487-496) (herein specifically incorporated by reference in itsentirety).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of mAbs directed against the same antigen according to thesimilarities of the binding profile of each antibody to chemically orenzymatically modified antigen surfaces (US 2004/0101920, hereinspecifically incorporated by reference in its entirety). Each categorymay reflect a unique epitope either distinctly different from orpartially overlapping with epitope represented by another category. Thistechnology allows rapid filtering of genetically identical mAbs, suchthat characterization can be focused on genetically distinct mAbs. Whenapplied to hybridoma screening, MAP may facilitate identification ofrare hybridoma clones that produce mAbs having the desiredcharacteristics. MAP may be used to sort the anti-TL1A mAbs of theinvention into groups of mAbs binding different epitopes.

The present invention includes hTL1A antibodies that bind to the sameepitope as any of the specific exemplary antibodies described herein.Likewise, the present invention also includes anti-hTL1A antibodies thatcompete for binding to hTL1A or a hTL1A fragment with any of thespecific exemplary antibodies described herein.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-hTL1A antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-hTL1Aantibody of the invention, the reference antibody is allowed to bind toa hTL1A protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the hTL1A molecule is assessed. Ifthe test antibody is able to bind to hTL1A following saturation bindingwith the reference anti-hTL1A antibody, it can be concluded that thetest antibody binds to a different epitope than the reference anti-hTL1Aantibody. On the other hand, if the test antibody is not able to bind tothe hTL1A molecule following saturation binding with the referenceanti-hTL1A antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-hTL1A antibody of theinvention.

To determine if an antibody competes for binding with a referenceanti-hTL1A antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to a hTL1A molecule under saturatingconditions followed by assessment of binding of the test antibody to thehTL1A molecule. In a second orientation, the test antibody is allowed tobind to a hTL1A molecule under saturating conditions followed byassessment of binding of the reference antibody to the TL1A molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the TL1A molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to hTL1A.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-TL1A monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxin agents include any agent that is detrimental tocells. Examples of suitable cytotoxin agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, see for example, WO05/103081, herein specifically incorporated by reference).

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al., 1991, J. Immunol. 147:60-69. The human anti-hTL1A mAbs can belinked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment, to produce abispecific or a multispecific antibody with a second bindingspecificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Bioequivalents

The anti-hTL1A antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described mAbs, but that retain the ability to bind humanTL1A. Such variant mAbs and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described mAbs. Likewise, the hTL1AmAb-encoding DNA sequences of the present invention encompass sequencesthat comprise one or more additions, deletions, or substitutions ofnucleotides when compared to the disclosed sequence, but that encode ananti-hTL1A antibody or antibody fragment that is essentiallybioequivalent to an anti-hTL1A antibody or antibody fragment of theinvention. Examples of such variant amino acid and DNA sequences arediscussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied. In one embodiment, two antigen-binding proteins arebioequivalent if there are no clinically meaningful differences in theirsafety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-hTL1A antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-hTL1A antibodies or antigen-binding fragments thereof of thepresent invention and the therapeutic methods using the same. Theadministration of therapeutic compositions in accordance with theinvention will be administered with suitable carriers, excipients, andother agents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA, 1998, JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, the purpose of the treatment, conditions,route of administration, and the like. When the antibody of the presentinvention is used for treating various conditions and diseases directlyor indirectly associated with TL1A, including inflammatorydiseases/disorders, autoimmune diseases/disorders, allergic reactions,and the like, in an adult patient, it is advantageous to intravenouslyor subcutaneously administer the antibody of the present invention at asingle dose of about 0.01 to about 20 mg/kg body weight, more preferablyabout 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3mg/kg body weight. Depending on the severity of the condition, thefrequency and the duration of the treatment can be adjusted. In certainembodiments, the antibody or antigen-binding fragment thereof of theinvention can be administered as an initial dose of at least about 0.1mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, orabout 10 to about 200 mg, to about 100 mg, or to about 50 mg. In certainembodiments, the initial dose may be followed by administration of asecond or a plurality of subsequent doses of the antibody orantigen-binding fragment thereof in an amount that can be approximatelythe same or less than that of the initial dose, wherein the subsequentdoses are separated by at least 1 day to 3 days; at least one week, atleast 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; atleast 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; atleast 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer, 1990, Science 249:1527-1533; Treat etal., 1989, in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 0.1 to about 800 mg per dosage form in aunit dose; especially in the form of injection, the aforesaid antibodyis contained in about 1 to about 500 mg, in about 5 to 300 mg, in about8 to 200 mg, and in about 10 to about 100 mg for the other dosage forms.

Combination Therapies

The invention further provides therapeutic methods for treating diseasesor disorders, which is directly or indirectly associated with hTL1A, byadministering the hTL1A mAb or fragment thereof of the invention incombination with one or more additional therapeutic agents. Theadditional therapeutic agent may be one or more of any agent that isadvantageously combined with the antibody or fragment thereof of theinvention, including immunosuppressants, anti-inflammatory agents,analgesic agents, anti-allergy agents, and the like. Suitableimmunosuppressants include, but are not limited to, glucocorticoids,cyclosporin, methotrexate, interferon β (IFN-β), tacrolimus, sirolimus,azathioprine, mercaptopurine, opioids, mycophenolate, TNF-bindingproteins, such as infliximab, eternacept, adalimumab, and the like,cytotoxic antibiotics, such as dactinomycin, anthracyclines, mitomycinC, bleomycin, mithramycin, and the like, antibodies targeting immunecells, such as anti-CD20 antibodies, anti-CD3 antibodies, and the like.Suitable anti-inflammatory agents and/or analgesics for combinationtherapies with the anti-hTL1A antibodies include, corticosteroids,non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin,ibuprofen, naproxen, Cox-2 inhibitors, and the like, TNF-α antagonists(e.g., Infliximab or REMICADE® by Centocor Inc.; golimumab by CentocorInc.; etanercept or ENBREL® by Amgen/Wyeth; adalimumab or HUMIRA® byAbbott Laboratories, and the like), IL-1 antagonists (e.g., IL-1-bindingfusion proteins, for example, ARCALYST® by Regeneron Pharmaceuticals,Inc., see U.S. Pat. No. 6,927,044; KINERET® by Amgen, and the like),IL-6 antagonists (e.g., anti-IL-6 receptor antibodies as disclosed inU.S. Pat. No. 7,582,298, and ACTEMRA® by Roche), acetaminophen,morphinomimetics, and the like. Suitable anti-allergy agents, which canblock the action of allergic mediators, or to prevent activation ofcells and degranulation processes, include antihistamines,glucocorticoids, epinephrine (adrenaline), theophylline, cromolyn sodiumand anti-leukotrienes, such as montelukast (SINGULAIR® by Merck) orzafirlukast (ACCOLATE® by AstraZeneca), as well as anti-cholinergics,decongestants, mast cell stabilizers, and other compounds that canimpair eosinophil chemotaxis.

The hTL1A mAb or fragment thereof of the invention and the additionaltherapeutic agent(s) can be co-administered together or separately.Where separate dosage formulations are used, the antibody or fragmentthereof of the invention and the additional agents can be administeredconcurrently, or separately at staggered times, i.e., sequentially, inappropriate orders.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human TL1A

VELOCIMMUNE™ mice were immunized with human TL1A, and the antibodyimmune response monitored by antigen-specific immunoassay using serumobtained from these mice. Anti-hTL1A antibody-expressing B cells wereharvested from the spleens of immunized mice shown to have elevatedanti-hTL1A antibody titers and were fused with mouse myeloma cells toform hybridomas. The hybridomas were screened and selected to identifycell lines expressing hTL1A-specific antibodies using assays asdescribed below. The assays identified several cell lines that producedchimeric anti-hTL1A antibodies designated as H2M1681N, H2M1704N,H2M1804N, H2M1805N, H2M1817N and H2M1818N. These antibodies were laterconverted to hIgG4 isotype by replacing the respective mouse constantregions with the hIgG4 amino acid sequence of SEQ ID NO:257, whichcontains a S108P mutation in the hinge region, and designated asH4H1681N, H4H1704N, H4H1804N, H4H1805N, H4H1817N and H4H1818N,respectively.

Human TL1A-specific antibodies were also isolated directly fromantigen-immunized B cells without fusion to myeloma cells, as describedin U.S. Pat. No. 7,582,298, which is hereby incorporated by reference inits entirety. Heavy and light chain variable regions were cloned togenerate fully human anti-hTL1A antibodies designated as H4H1719P,H4H1725P, H4H1738P, H4H1742P, H4H1745P, H4H1750P and H4H1752P. Stablerecombinant antibody-expressing CHO cell lines were established.

Example 2 Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each Heavy Chain VariableRegion (HCVR) and Light Chain Variable Region (LCVR). Table 1 shows thegene usage for selected antibodies in accordance with the invention.

TABLE 1 HCVR LCVR Antibody V_(H) D_(H) J_(H) V_(K) J_(K) H2M1704 3-7 1-7 6 4-1  3 H2M1681 3-23  2-21 4 1-5  1 H2M1817 4-34 3-9 4 3-20 4H2M1804 4-34 1-1 4 3-20 4 H2M1818 3-11  4-17 6 4-1  1 H2M1805 4-34 3-3 42-24 4 H4H1719 3-9  3-3 6 2-28 2 H4H1725 1-2   2-15 3 1-12 4 H4H17383-15 4-4 6 2-28 2 H4H1742 3-23 2-2 6 1-9  2 H4H1745 3-23 6-6 4 1-9  4H4H1750 3-30  4-17 6 1-17 1 H4H1752 3-23 1-7 4 1-5  1

Table 2 shows the heavy and light chain variable region amino acidsequence pairs of selected anti-hTL1A antibodies and their correspondingantibody identifiers. The N and P designations refer to antibodieshaving heavy and light chains with identical CDR sequences but withsequence variations in regions that fall outside of the CDR sequences(i.e., in the framework regions). Thus, N and P variants of a particularantibody have identical CDR sequences within their heavy and light chainvariable regions but contain modifications within the framework regions.

TABLE 2 mAb Name HCVR/LCVR (H2M- or H4H-) SEQ ID NOS 1704N  2/10 1681N18/26 1804N 34/42 1805N 50/58 1817N 66/74 1818N 82/90 1719N  98/1061719P 114/116 1725N 118/126 1725P 134/136 1738N 138/146 1738P 154/1561745N 158/166 1745P 174/176 1750N 178/186 1750P 194/196 1752N 198/2061752P 214/216 1742N 218/226 1742P 234/236

Example 3 TL1A Binding Affinity Determination

Binding affinities and kinetic constants were determined by surfaceplasmon resonance at 25° C. and 37° C. as indicated in Tables 3-5 forhuman monoclonal anti-TL1A antibodies binding to the following TL1Aspecies variants: human (h) (CHO-expressed, residues 72-251 of SEQ IDNO:244, with N-terminal His₆-tag), cynomolgus monkey (Mf) (E.coli-expressed, residues 72-251 of SEQ ID NO:248, with or withoutN-terminal Met), cynomolgus monkey (CHO-expressed, residues 72-251 ofSEQ ID NO:248, with N-terminal His₆-tag), mouse (m) (E. coli-expressed,residues 76-252 of SEQ ID NO:250, with or without N-terminal Met), mouse(CHO-expressed, residues 76-252 of SEQ ID NO:250, with N-terminalHis₆-tag), and rat (CHO cell-expressed; residues 76-252 of SEQ IDNO:258, with N-terminal His₆-tag). Binding constants were alsodetermined for the hTL1A variant, Fhm (E. coli-expressed, residues72-251 of SEQ ID NO:246 containing Q167R substitution, with or withoutN-terminal Met). Measurements were conducted on a T100 BIACORE™instrument. Antibodies, expressed with either mouse Fc (designated withprefix “H2M”) or human IgG4(S108P) Fc (designated with prefix “H4H”),were captured onto an anti-Fc sensor surface, and at least threedifferent concentrations of the soluble TL1A proteins ranging from 1.25nM to 100 nM were injected over the sensor surface. Kinetic association(k_(a)) and dissociation (k_(d)) rate constants were determined byfitting the data to a 1:1 binding model using BIAevaluation 4.1 curvefitting software (BIAcore Life Sciences). Molar concentrations ofTL1A/Fhm used in the data fitting assumed a monomeric state for TL1A insolution. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (t_(1/2)) were calculated from the kinetic rateconstants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min)=[In2/(60*k_(d))].NB: No binding under the conditions tested; NT: Not tested in thisexperiment; *: Fitted k_(d) values below 1×10⁻⁶ (1/s) are slower thanthe detection limit under these experimental conditions; therefore,k_(d) values were set at 1×10⁻⁶ (1/s) for the purpose of approximatingK_(D) and t_(1/2); **: Equilibrium dissociation constants for antibodieswere determined under steady state conditions.

As shown in Tables 3 and 4, antibodies bound with high affinity toCHO-expressed forms of both human and monkey TL1A proteins at 25° C. (13and 12 antibodies with K_(D)<1 nM, respectively) and at 37° C. (13 and12 antibodies with K_(D)<1 nM, respectively). H4H1750P boundsignificantly weaker to the monkey compared to the human TL1A protein.H4H1704N bound to CHO-expressed mTL1A with K_(D)<2 nM at both 25° C. and37° C. H4H1818N bound CHO-expressed mTL1A at 25° C. (K_(D)˜7 nM) but notat 37° C. Five antibodies, H4H1681N, H4H1738P, H4H1750P, H4H1752P andH4H1805N, did not bind to CHO-expressed rat TL1A at either 25° C. or 37°C.; the other eight antibodies bound to rat TL1A at both temperatureswith K_(D) ranging from ˜0.6 pM to ˜16 nM.

As shown in Table 5, three antibodies (H2M1681N, H4H1752P and H2M1805N)did not demonstrate binding to the E. coli-expressed Fhm variant[hTL1A(Q167R)] under the conditions tested. Three antibodies (H2M1704N,H4H1725P, and H2M1818N) demonstrated weak binding (K_(D) ranging from˜60 nM to ˜170 nM) to mouse TL1A expressed in E. coli as assessed understeady-state conditions, while all other tested antibodies did not bindto the mouse TL1A protein under the tested conditions.

TABLE 3 CHO-expressed TL1A at 25° C. hTL1A hTL1A MfTL1A MfTL1A mTL1AmTL1A rTL1A rTL1A K_(D) t_(1/2) K_(D) t_(1/2) K_(D) t_(1/2) K_(D)t_(1/2) mAb (pM) (min) (pM) (min) (pM) (min) (pM) (min) H4H1681N 263  36404 25 NB NB NB NB H4H1704N 39.2 453 59.9 276 194 46 404 25 H4H1719P 481 44 417 45 NB NB 364 38 H4H1725P 63.6 185 346 64 NB NB 317 26 H4H1738P608  64 361 93 NB NB NB NB H4H1742P 60.4 755 115 577 NB NB  78 144 H4H1745P 164 172 115 231 NB NB 2.7 (nM)  4 H4H1750P 15.8* 11550*  8.6(nM) 21 NB NB NB NB H4H1752P 156 197 213 139 NB NB NB NB H4H1804N 291 51 264 49 NB NB 321 43 H4H1805N 365  73 342 74 NB NB NB NB H4H1817N 321103 356 92 NB NB 2.5 (nM) 25 H4H1818N 124 120 119 122 7.1 (nM) 12  88 92

TABLE 4 CHO-expressed TL1A at 37° C. hTL1A hTL1A mTL1A rTL1A K_(D)t_(1/2) MfTL1A MfTL1A mTL1A t_(1/2) rTL1A t_(1/2) mAb (pM) (min) K_(D)(pM) t_(1/2) (min) K_(D) (pM) (min) K_(D) (pM) (min) H4H1681N 254  31226  32 NB NB NB NB H4H1704N 1.00* 11550* 0.93* 11550* 1.3 27 46 133 (nM) H4H1719P 7.61 1912 2.01 5784 NB NB 78 86 H4H1725P 23.9  758 17.7 888 NB NB 411 15 H4H1738P 571  51 465  60 NB NB NB NB H4H1742P 4.37*11550* 4.45* 11550* NB NB 653 27 H4H1745P 177  129 173  124 NB NB 16.5(nM) 11 H4H1750P 13.8* 11550* 17.1  11 NB NB NB NB (nM) H4H1752P 225  96223  91 NB NB NB NB H4H1804N 45.8  286 78  167 NB NB 127 88 H4H1805N 299 80 352  68 NB NB NB NB H4H1817N 27.8 1098 25.6 1150 NB NB  1.6 (nM) 26H4H1818N 0.925* 11550* 0.804* 11550* NB NB 0.61* 11550* 

TABLE 5 E. coli-expressed Fhm/TL1A 25° C. 37° C. 25° C. 37° C. 25° C.Fhm Fhm Fhm Fhm MfTL1A MfTL1A MfTL1A MfTL1A mTL1A mAb K_(D) (pM) t_(1/2)(min) K_(D) (pM) t_(1/2) (min) K_(D) (pM) t_(1/2) (min) K_(D) (pM)t_(1/2) (min) K_(D) (pM)** H2M1681N NB NB NT NT 546 54 1.5 (nM) 14 NBH2M1704N 282 52 NT NT 285 57 751 16 127 (nM) H4H1719P 109 96 174 42 24279 289 40 NB H4H1725P 28.9 447 43.6 194 63.7 292 62.2 174  62 (nM)H4H1738P 1020 19 3100 4 1360 18 4.2 (nM) 4 NB H4H1742P 437 129 696 45593 97 945 35 NB H4H1745P 115 226 207 71 141 221 281 74 NB H4H1750P 204623 244 335 52.7 (nM) 4 456 (nM)  1 NB H4H1752P NB NB NB NB 274 122 132029 NB H2M1804N 192 108 NT NT 176 172 218 118 NB H2M1805N NB NB NT NT 34567 439 36 NB H2M1817N 451 57 NT NT 1250 37 1.0 (nM) 30 NB H2M1818N 108017 NT NT 1840 10 4.1 (nM) 3 171 (nM)

Experiment 4 Inhibition of TL1A by Anti-hTL1A Antibodies

HEK293 cell lines (CRK01573, ATCC) were generated to stably expresshuman DR3 (full-length; SEQ ID NO:252) or mouse DR3 (full-length; SEQ IDNO:259) along with a luciferase reporter [NFκB response element(5×)-luciferase-IRES-GFP]. NFκB activation by TL1A has been shownpreviously (Migone et al., 2002, Immunity 16:479-492). In order to testthe membrane-bound form of TL1A and TL1A variants, HEK293 cell lineswere generated that stably express full length human TL1A (SEQ IDNO:244), full-length human TL1A with Gln-167 substituted by Arg [Fhm;TL1A(Q167R); SEQ ID NO:246], full-length TL1A from cynomolgus monkey,Macaca fascicularis (MfTL1A; SEQ ID NO:248), full length mouse TL1A (SEQID NO:250), and full length rat TL1A (SEQ ID NO:258). The stable celllines were isolated and maintained in 10% fetal bovine serum (FBS;Irvine Scientific), Dulbecco's Modified Eagle Medium (DMEM; IrvineScientific), non-essential amino acids (NEAA; Irvine Scientific),Penicillin/Streptomycin (Invitrogen), and G418 (Invitrogen).

For the bioassay, human or mouse DR3 reporter cells were seeded into96-well assay plates at 1×10⁴ cells/well in low serum media, i.e., 0.1%FBS and OPTIMEM® (Invitrogen), and incubated at 37° C. and 5% CO₂overnight. The next day, soluble TL1A or FHM (sTL1A or sFHM) wasserially diluted at 1:3 and added to cells at concentrations rangingfrom 0.002 nM to 100 nM (plus a buffer control containing no TL1A). Forinhibition, antibodies were serially diluted at 1:3 and added to cellsat concentrations ranging from 0.002 nM to 100 nM (plus a buffer controlcontaining no antibody) in the presence of constant concentrations ofTL1A or Fhm: 800 pM hTL1A (CHO cell-expressed; residues 72-251 of SEQ IDNO:244, with N-terminal His₆-tag), 100 pM hTL1A (E. coli-expressed;residues 72-251 of SEQ ID NO:244, with or without N-terminal Met), 500pM Fhm (CHO cell-expressed; residues 72-251 of SEQ ID NO:246), 400 pMMfTL1A (CHO cell-expressed; residues 72-251 of SEQ ID NO:248, withN-terminal His₆-tag), 400 pM MfTL1A (E. coli-expressed; residues 72-251of SEQ ID NO:248, with or without N-terminal Met), 50 pM mouse TL1A (CHOcell-expressed; residues 76-252 of SEQ ID NO:250, with N-terminalHis₆-tag), 20 pM mouse TL1A (E. coli-expressed; residues 76-252 of SEQID NO:250, with or without N-terminal Met), and 50 pM rat TL1A (CHOcell-expressed; residues 76-252 of SEQ ID NO:258, with N-terminalHis₆-tag). Luciferase activity was detected after 5.5 hours ofincubation at 37° C. and 5% CO₂. The results are shown in Table 6.Control mAb1: Positive control (an anti-hTL1A antibody with heavy andlight chain variable domains having the amino acid sequencescorresponding to SEQ ID NOS:21 and 27 of US 2009/0280116); control mAb2:Negative control (irrelevant antibody); NB: No binding under theconditions tested; NT: Not tested in this assay; *: Inhibition is not tobaseline at the highest antibody concentration of 100 nM.

TABLE 6 hTL1A hTL1A Fhm MfTL1A MfTL1A mTL1A mTL1A rTL1A sTL1A or sFhm(CHO) (E. Coli) (CHO) (CHO) (E. Coli) (CHO) (E. Coli) (CHO) EC50 (nM)0.63 0.12 0.32 0.86 2.02 0.08 0.01 0.06 Constant TL1A or 800 100 500 400400 50 20 50 Fhm (pM) IC50 H4H1681N 0.17 0.02 NB 0.03 0.02 NB NB NB [nM]H4H1704N 0.13 0.06 0.17 0.01 0.03 NB NB 0.40 H4H1804N 0.07 0.03 0.100.03 0.02 NB NB 3.64 H4H1805N 0.10 0.04 185.50* 0.03 0.02 NB NB NBH4H1817N 0.11 0.04 0.12 0.04 0.02 NB NB 23.87 H4H1818N 0.37 0.29 0.620.13 0.06 NB NB 1.10 H4H1719P 0.06 0.02 0.08 0.01 0.02 NT NB NT H4H1725P0.05 0.02 0.07 0.01 0.02 NB NB 63.43 H4H1738P 0.39 0.16 0.33 0.39 0.07NT NB NT H4H1742P 0.31 0.19 0.53 0.26 0.07 NB NB 6.12 H4H1745P 0.09 0.060.15 0.05 0.03 NB NB NB H4H1750P 0.90 2.17 3.10 154.70 32.47* NT NB NTH4H1752P 0.36 0.21 NB 0.12 0.05 NT NB NT Control NB 0.74 NB NB 3.25 NTNB NT mAb1 Control NB NB NB NB NB NB NT NB mAb2

As shown in Table 6, thirteen anti-TL1A antibodies were shown to inhibitsoluble human TL1A (CHO and E. coli-expressed) stimulation of the humanDR3 receptor expressed on HEK293 cells as determined using a luciferasereporter for NFκB activation. A positive control antibody (control mAb1)inhibited E. coli-expressed, but not CHO-expressed, hTL1A. Tenantibodies also inhibited stimulation of hDR3-expressing cells by Fhm(hTL1A with Q167R). H4H1681N, H4H1805N and H4H1752P did not fullyinhibit Fhm at the highest antibody concentration of 100 nM. Allthirteen antibodies also blocked MfTL1A (Table 6). Mouse TL1A (producedfrom both CHO and E. coli) stimulated NFκB activation in the hDR3reporter cells; however, none of the 13 anti-human TL1A antibodiesinhibited E. coli-expressed mouse TL1A in this assay (Table 6). Nineselected antibodies were further tested and did not demonstrate blockingof CHO-expressed mouse TL1A in this assay (Table 6).

To test the ability of TL1A expressed on cells to stimulate signaling inthe hDR3 reporter system, bioassays were performed as described abovefor soluble TL1A with the following changes: Adherent HEK293/TL1A cellswere dissociated using Enzyme-Free Dissociation Solution (Chemicon) andadded to adherent hDR3 reporter cells after serially diluting the TL1Acells at 1:2 starting from 2×10⁵ cells to 195 cells (plus a no-cellcontrol). For inhibition by antibodies, 1×10⁴ cells were added togetherwith serially diluted antibodies from 100 nM to 0.002 nM (plus a controlcontaining no antibody). The results are shown in Table 7. Control mAb1and mAb2: Same as the assays above. NB: No binding under the conditionstested; NT: Not tested in this assay; *: Inhibition is not to baselineat the highest antibody concentration of 100 nM.

TABLE 7 Cell-Bound TL1A or HEK293/ HEK293/ HEK293/ HEK293/ HEK293/ FhmhTL1A Fhm MfTL1A mTL1A rTL1A EC50 (cells) 23474 47921 8465 12366 9773Constant TL1A or 10,000 Fhm (# cells) IC50 [nM] H4H1681N 0.66 NB 3.07 NTNT H4H1704N 1.11 1.58 3.76 NB NT H4H1804N 0.56 1.23 2.23 NB 3.99H4H1805N 0.82 54.10* 1.93 NB NB H4H1817N 1.11 0.62 5.04 NB 94.96*H4H1818N 1.54 1.42 4.29 NT NT H4H1719P 0.82 0.84 3.00 NT NT H4H1725P0.66 0.94 2.82 NB 190.30* H4H1738P 7.47 7.75 20.25 NT NT H4H1742P 6.558.39 18.45 NB NT H4H1745P 0.76 2.12 4.28 NT NT H4H1750P 12.82 29.52*107.40* NT NT H4H1752P 1.99 138.10* 11.67 NT NT Control mAb1 NB NB NB NBNT Control mAb2 NB NB NB NB NB

As shown in Table 7, all thirteen antibodies blocked the stimulation ofhDR3-expressing cells by hTL1A expressed on cells. With cell-bound Fhm,all antibodies inhibited significantly except H4H1681N, H4H1805N,H4H1750P and H4H1752P, which did not inhibit fully at the highest testedantibody concentration of 100 nM. With cell-bound MfTL1A, all antibodiesinhibited, except H4H1750 that did not inhibit completely at the highesttested antibody concentration of 100 nM. Six of the antibodies H4H1704N,H4H1804N, H4H1805N, H4H1817N, H4H1725P, and H4H1742P were tested forblocking cell-surface mTL1A stimulation of mDR3 cells; and none showedinhibition. Reporter cells expressing mouse DR3 could also be stimulatedby rTL1A-expressing 293 cells, with an observed EC₅₀ of 9773 cells(Table 7). Four antibodies were tested in the rTL1A/mDR3 assay: threeantibodies H4H1804N, H4H1817N, H4H1725P blocked while H4H1805N did notblock stimulation of mDR3 cells by cell-surface rTL1A (Table 7). ControlmAb1 blocked E. coli-expressed soluble hTL1A and MfTL1A stimulation ofhDR3 cells, but failed to block the CHO-expressed forms of hTL1A andMfTL1A under all tested conditions (Table 6). Control mAb1 also did notinhibit stimulation of hDR3-expressing cells by any of the cell-surfaceexpressed TL1A and Fhm under all tested conditions (Table 7).

Experiment 5 Blocking of TL1A to hDR3 and DcR3 by Anti-TL1A Antibodies

The ability of antibodies to block human TL1A binding to its cognatereceptors, the DR3 and DcR3 receptors, was measured using a competitionsandwich ELISA. In addition, blocking of a human TL1A variant FHM (humanTL1A Q167R) and the cynomolgus monkey (Macaca fascicularis) TL1A(MfTL1A) protein binding to the human DR3 or DcR3 receptors was measuredin the same manner. Constant amounts of biotinylated human TL1A or FHM(both expressed with a 6-His tag in CHO cells) or biotinylated MfTL1A(expressed in CHO cells) were separately titrated with varying amountsof antibodies. The antibody-protein complexes were incubated in solution(1 hr, 25° C.) and then transferred to microtiter plates coated withhuman DR3 (hDR3) or human DcR3 (hDcR3) expressed as human IgG1 Fc fusionproteins. After one hour at 25° C. the wells were washed, and boundhuman or monkey TL1A was detected with streptavidin conjugated withhorseradish peroxidase (HRP). Wells were developed with a TMB solutionto produce a colorimetric reaction and quenched with aqueous sulfuricacid before reading absorbance at 450 nm on a Perkin-Elmer Victor X5plate reader. A sigmoidal dose-response curve was fit to the data usingthe Prism™ data analysis package. The calculated IC50 value, defined asthe concentration of antibody required to block 50% of TL1A binding tohDR3 or hDcR3, was used as an indicator of blocking potency. Both fullyhuman anti-hTL1A mAbs and comparator antibodies, i.e., control mAb1 (ananti-hTL1A antibody with heavy and light chain variable domains havingthe amino acid sequences corresponding to SEQ ID NOS:21 and 27,respectively, of US 2009/0280116) and control mAb3 (an anti-hTL1Aantibody with heavy and light chain variable domains having the aminoacid sequences corresponding to SEQ ID NOS:57 and 48, respectively, ofUS 2009/0280116), were included in the study. The results are shown inTable 8. NB: No binding under the conditions tested; NT: Not tested.Concentrations of biotinylated soluble ligands: (1) 150 pM; (2) 500 pM;(3) 10 pM; and (4) 50 pM.

As shown in Table 8, most of the fully human mAbs show effectiveblocking of the TL1A/hDR3 and TL1A/hDcR3 binding interaction, withseveral showing IC50 values below 50 pM. Two of the antibodies, H4H1752Pand H4H1805N, strongly blocked binding of both human and monkey TL1Abinding to both hDR3 and hDcR3 but failed to block binding of FHM toeither hDR3 or hDcR3, suggesting that the binding epitope for these twoantibodies may involve the region near the FHM mutation site (hTL1A withQ167R). The crystal structure of hTL1A shows that residue Q167 occurswithin a surface-exposed loop (Zhan et al., 2009, Biochemistry 48:7636-7645).

TABLE 8 DcR3 hDR3 hDR3 hDR3 DcR3 DcR3 MfTL1A hTL1A (CHO)¹ FHM (CHO)¹MfTL1A (CHO)² hTL1A (CHO)³ FHM (CHO)³ (CHO)⁴ mAb ID IC₅₀ (pM) IC₅₀ (pM)IC₅₀ (pM) IC₅₀ (pM) IC₅₀ (pM) IC₅₀ (pM) H4H1681N 60 >10000 141 17 90 93H2M1681N 37 >10000 61 13 234 149 H4H1704N 30 44 42 77 170 110 H2M1704NNT NT NT NT NT NT H4H1719P 22 23 46 44 45 61 H4H1725P 15 18 16 68 85 145H4H1738P 69 152 117 122 150 68 H4H1742P 64 214 240 181 231 127 H4H1745P18 44 50 58 85 118 H4H1750P 341 589 5209 656 626 NB H4H1752P 104 NB 11031 NB 56 H4H1804N 40 69 71 175 >10000 34 H2M1804N 46 102 81 120 >10000 9H4H1805N 14 NB 26 33 436 313 H2M1805N 6 NB 13 12 2241 1138 H4H1817N 114235 101 270 NT 322 H2M1817N 154 249 137 890 666 776 H4H1818N 119 202 123232 NT 1102 H2M1818N 154 317 239 396 NB 55 Control >10000 NB 5300 >1000NT 21000 mAb1 Control >10000 NB 17000 8600 NT NT mAb3

Example 6 Cell Surface Binding Competition of Anti-TL1A Antibodies withSoluble hTL1A

Human embryonic kidney 293 cells stably transfected to over-expresscell-surface hTL1A were first stained in a flow cytometric experimentwith eight anti-hTL1A antibodies at four concentrations (1, 0.1, 0.01,and 0.003 μg/ml). Bound human antibodies were detected using anallophycocyanin-labeled goat F(ab′)₂ specific for human Fcγ [oranti-hFcγ-APC F(ab′)₂, Jackson ImmunoResearch, #109-136-170]. The lowestantibody concentration providing significant staining levels was thenused in a competition binding experiment. A negative isotype controlantibody (human IgG4) was used at 1 μg/ml to define the backgroundsignal. For the competition experiment, eight antibody samples, at theminimal concentrations identified above, were first treated with solublehTL1A expressed from CHO cells at concentrations ranging from 0.03 μg/mlto 10 μg/ml. After pre-incubation for 30 min on ice, the antibody/hTL1Amixture was added to 293/HEK-hTL1A cells that had been isolated bycentrifugation in a 96-well conical plate. After incubation for anadditional 10 minutes on ice, the cells were washed. The secondaryreagent, anti-h Fcγ-APC F(ab′)₂, was added to all wells at a 200-folddilution to detect bound antibodies. Samples were incubated for 15minutes on ice, away from light, and then washed. Cells were processedon an BD™ LSR II Flow Cytometer (BD Biosciences) to detect anti-hTL1Aantibodies bound to the cell surface, and data were analyzed usingFlowJo software (version 8.8.6; Tree Star Inc.). The results are shownin Table 9. Maximum signal: Anti-hTL1A antibody binding in the absenceof soluble hTL1A; Minimum signal: Signal recorded when 1 μg/ml ofisotype control antibody was added in place of the anti-hTL1A antibody.NT: Not tested.

TABLE 9 Mean Fluorescence Intensity for Soluble anti-hTL1A antibodies(H4H) binding to cell-surface hTL1A hTL1A 1704N 1725P 1742P 1804N 1805N1817N 1681N 1745P (μg/ml) 0.1 μg/ml 0.1 μg/ml 1 μg/ml 0.1 μg/ml 0.1μg/ml 1 μg/ml 1 μg/ml 1 μg/ml 10 NT NT NT NT NT NT 16.9 15 3 22.6 34.919.9 28.9 23.6 25.2 28.5 19.5 1 26 29.2 32.7 33.7 25.1 29.1 80.9 26.90.3 31.5 40.7 44.7 33.5 36.8 23.6 236 115 0.1 132 84.5 79.4 51.1 60.297.2 327 93.1 0.03 163 207 126 126 156 85.3 318 80 Maximum 116 211 126127 158 110 320 87.2 signal Minimum 27.5 27.5 27.5 27.5 27.5 27.5 17.917.9 signal

As shown in Table 9, the signals from the eight tested antibodies couldbe competed down to baseline levels by the addition of excess solublehTL1A, demonstrating the specificity of binding of the antibodies tocell-surface hTL1A.

Example 7 Blocking of hTL1A-Dependent CD4⁺ T-Cell Stimulation byAnti-TL1A Antibodies

To determine the ability of anti-hTL1A antibodies to blockhTL1A-dependent stimulation of human CD4⁺ T-cells, an in vitro assay wasdeveloped in which hTL1A/anti-CD3/anti-CD28-stimulated release ofIFN-gamma (IFN-γ) was measured in the presence or absence of antibodies.Human CD4⁺ T-cells were isolated from fresh buffy coats prepared fromhuman blood samples obtained from the New York Blood Center. Cells froma single donor were kept separate from other donor cells for each assay.The CD4⁺ T-cells were added to the wells of a 96-well plate at 3.5×10⁵cells per well. To each well was then added soluble hTL1A (residues72-251 of NP_005109.2 with an N-terminal hexa-histidine tag, expressedfrom CHO cells) to a final concentration of 1 μg/ml (16 nM, assuminghTL1A forming trimers in solution) in RPMI+10% FBS, L-glutamine andpenicillin/streptomycin. To each well was also added the anti-hTL1Aantibodies or an isotype control antibody to final concentrations of 1.0μg/ml or 3.0 μg/ml (6.7 nM or 20 nM, respectively). The samples wereincubated for 15 minutes at 4° C. in the dark, followed by the additionsof anti-hCD3 (BD Pharmingen, cat #555336) and anti-hCD28 (BD Pharmingen,cat #555725) to each well to final concentrations of 1.0 μg/ml. Sampleswere incubated for 24 hours at 37° C., the supernatants harvested, andIFN-γ levels determined by ELISA. The blocking effect (average from twoseparate wells for each condition) of each antibody on each human CD4⁺T-cell donor sample is represented as the reduction from maximal signaldivided by maximal response window; i.e., %Blocking=[(Max−Inhib)/(Max−Min)]×100, where “Max”, “Inhib”, and “Min”are concentrations of IFN-γ measured for CD4⁺ human T-cells treated asfollows: “Max”—treated with [hTL1A+anti-hCD3+anti-hCD28+isotype controlmAb]; “Min:”—treated with [anti-hCD3+anti-hCD28+isotype control mAb];and “Inhib”—treated with [hTL1A+anti-hCD3+anti-hCD28+anti-hTL1A testmAb]. Antibodies for which IFN-γ blockade surpassed the “Min” baselinelevel are represented as 100% blockade. Ratio (Max/Min) is the ratio ofthe IFN-γ concentration produced from human CD4⁺ T-cells treated underMax and Min conditions as defined above.

As shown in Table 10, the antibodies H4H1725P, H4H1805N, H4H1817N, andH4H1804N significantly blocked hTL1A-stimulated IFN-γ release at both 1μg/ml and 3 μg/ml concentrations, with nearly complete blockade (>80%)observed for most donors at the higher antibody concentration. Theresults for blockade of IFN-γ secretion by thirteen different anti-hTL1Aantibodies against CD4⁺ T-cells from 10 different human donors arefurther summarized in Table 11. SD: Standard Deviation.

TABLE 10 % Blocking of IFN-γ production in human T-cells from 10 donorsDonor # Ratio D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 mAb ID (Max/Min) 5 4 10 104 3 4 3 8 2 H4H1725P mAb 90 100 70 85 45 80 60 85 50 95 H4H1805N 1 μg/ml100 100 90 100 100 100 100 100 90 100 H4H1817N (6.7 nM) 100 100 90 90100 45 55 100 55 80 H4H1804N 95 100 80 90 100 50 10 100 50 0 H4H1725PmAb 95 100 95 100 90 80 90 100 90 100 H4H1805N 3 μg/ml 100 100 95 100 80100 100 100 70 100 H4H1817N (20 nM) 100 100 100 100 100 100 100 100 9585 H4H1804N 100 100 95 100 100 100 100 100 100 80

TABLE 11 Average % Average % Average % Blocking (SD) Blocking (SD)Blocking (SD) mAb ID 0.1 μg/ml mAb 1 μg/ml mAb 3 μg/ml mAb H4H1681N 20%(24) 45% (30) 95% (7) H4H1704N 22% (29) 53% (27) 98% (5) H4H1719P 10%(22) 37% (23) 78% (30) H4H1725P 13% (14) 80% (20) 94% (7) H4H1738P 25%(30) 41% (35) 81% (18) H4H1742P 10% (22) 58% (33) 83% (16) H4H1745P 22%(27) 36% (36) 91% (7) H4H1750P 11% (14) 42% (35) 89% (15) H4H1752P 18%(25) 52% (35) 71% (30) H4H1804N 26% (30) 68% (38) 98% (6) H4H1805N 21%(28) 98% (4)  94% (10) H4H1817N 25% (33) 81% (22) 98% (5) H4H1818N 25%(33) 42% (33) 71% (35) Isotype Control 16% (21) 26% (33) 28% (27)

IFN-γ levels were also measured at six different antibody concentrations(ranging from 0.03 μg/ml to 10 μg/ml) for each of six differentantibodies added to CD4⁺ T-cells from twelve human donors. Curve fittingto the data allowed estimation of the antibody concentration at whichhalf-maximal inhibition was achieved for each antibody for each donorcell sample. The average (±SD) concentrations for achieving half-maximalinhibition are provided in Table 12.

TABLE 12 mAb IC₅₀ (nM) Donor # H4H1725P H4H1742P H4H1805N H4H1817NH4H1804N H4H1704N D1 3.4 24 2.4 6.6 6.8 — D2 3.2   4.6 5.5 3.1 8.6 — D35.4 10 3.4 4.7 8.6 — D4 4.7 13 2.5 2.4 7.7 — D5 7.0 12 2.9 8.5 6.7 17 D65.4 10 3.4 4.7 8.6 11 D7 13 31 8.0 7.0 12 12 D8 12 27 5.1 7.0 11 19 D96.4 56 5.9 9.5 8.1 7.5 D10 4.1 12 2.8 7.3 12 10 D11 6.1 27 3.0 7.3 7.611 D12 6.4 14 3.1 9.4 7.5 8.0 Average 6.4 20 4.0 6.5 8.8 12 (±SD) (3.1)(14) (1.7) (2.3) (1.9) (4.1)

Four of the antibodies, H4H1725P, H4H1804N, H4H1805N, H4H1817N exhibitedaverage half-maximal inhibition concentrations below 10 nM (rangingapproximately 4-9 nM).

The invention claimed is:
 1. A method for treating a disease or disorderwhich is ameliorated, improved or inhibited by blocking or inhibitingTL1A activity, comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising an isolated human antibody or antigen-binding fragmentthereof that specifically binds human TNF-like ligand 1A (hTL1A),comprising a heavy chain complementarity determining region 1 (HCDR1),HCDR2 and HCDR3 and a light chain complementarity determining region 1(LCDR1), LCDR2 and LCDR3 sequence combination of HCDR 1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3, selected from SEQ ID NO:4/6/8/12/14/16,20/22/24/28/30/32, and 52/54/56/60/62/64, and a pharmaceuticallyacceptable carrier.
 2. The method according to claim 1, wherein thedisease or disorder is selected from ulcerative colitis, Crohn'sdisease, rheumatoid arthritis, multiple sclerosis, asthma and allergiclung inflammation.
 3. The method according to claim 2, furthercomprising administering one or more additional therapeutic agentsselected from an immunosuppressant, an anti-inflammatory agent, ananalgesic, and an anti-allergy agent.
 4. The method according to claim1, wherein the heavy and light chain CDR sequences are selected from:(i) SEQ ID NO: 4/6/8/12/14/16 and (ii) SEQ ID NO: 20/22/24/28/30/32. 5.The method according to claim 4, wherein the antibody or antigen-bindingfragment comprises a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO: 2 or 18, respectively.
 6. The methodaccording to claim 4, wherein the antibody or antigen-binding fragmentcomprises a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO: 10 or 26, respectively.
 7. The method accordingto claim 1, wherein the antibody or fragment comprises an HCVR/LCVRsequence pair selected from SEQ II) NO: 18/26 or 50/58, and wherein theantibody or fragment does not cross-react with Fhm comprising the aminoacid sequence of SEQ ID NO: 246.